The present invention relates generally to the introduction of foreign genes into mammalian cells. More specifically, it relates to a new and improved carrier system and method for the non-invasive targeted delivery of soluble nucleic acid complexes to such cells.
The introduction of foreign genes into mammalian cells in vitro is frequently used for studying gene regulation. Various techniques for accomplishing this have been employed heretofore. The most popular method for this gene transformation employs a precipitation technique in which calcium phosphate is used as a coprecipitate with DNA to form insoluble particles. These particles, or at least some of them, become internalized within the host cells by endocytosis, resulting in the expression of the new or exogenous genes. This internalization is non-specific with respect to the cells of particular organs and does not rely upon any particular recognition site for the endocytosis. Although this technique is widely applicable in vitro, its use in vitro is limited by a lack of cell specificity, relatively low efficiency especially in certain highly differentiated cells. In addition, its use in vivo would be limited by the insoluble nature of the resulting precipitates. Other techniques also have problems. For example, viruses that have foreign genes incorporated within the viral DNA could be used either in vitro or in vivo. However, since the viral genes are also delivered simultaneously with the desired new gene, undesirable viral effects may be produced. As with the calcium phosphate precipitation technique, the use of viral agents is not generally specific for particular cell types.
Gene delivery also has been accomplished experimentally both in vitro and in vivo by using liposome entrapped DNA. Liposomes are small membrane-enclosed spheres that have been formed with the appropriate DNA trapped within. However, this system also has inherent problems. It is difficult to control the size of the liposome and, hence the uniformity of delivery to individual cells. Additionally, it is difficult to prevent leakage of the contents of the liposomes and, as with the other techniques, there has been difficulty in directing cell-type specificity. Electroporation and DEAE-dextran techniques are toxic to cells although a proportion of cells that survive are transformed.
Accordingly, it is an object of the present invention to provide a new and improved carrier system that can introduce foreign genes in a soluble non-toxic, cell-specific manner into mammalian cells. Included in this object is the provision for a system and method that exhibits a high degree of cell specificity using a relatively simple yet reliable delivery or carrier system. The solubility of the carrier system is a significant feature since it provides the possibility of targeting genes in vitro to a specific cell type present in a mixture of cell types. The present invention permits this without the necessity for simultaneously delivering viral genes and the undesirable viral effects that may be produced as a result thereof.
Another feature of the present invention is the use of receptor-mediated endocytosis to endow cell specificity to the gene delivery system. This involves the use of cell-surface receptors as naturally existing entry mechanisms for the specific delivery of genes. Included in this feature is the provision for a unique soluble polynucleotide complex capable of targeting the gene to specific cells possessing particular receptors that recognize the complex. The complex, in turn, employs a ligand conjugate as a new and improved carrier for DNA that can direct the complex to specific cell types having appropriate receptors.
A special feature of the present invention is the utilization of a specific type of non-covalent linkage provided by a component of the conjugate for binding polynucleotides electrostatically to the conjugate in a non-damaging fashion. An additional feature is the coupling of the polynucleotide binding component to a ligand by a bond that can result in facile cleavage from the ligand following internalization.
These features and advantages of the present invention all directly contribute to the efficiency and targetability to specific cell types, including normal cells as well as hepatoma cell systems, and form the basis for in vitro delivery in an efficient and effective manner while avoiding the disadvantages of prior systems. The provision of a soluble gene delivery system is particularly attractive for the study of how genes are controlled in vitro and in vivo. In addition, by virtue of its solubility and targetability, this system may have potential for replacement of defective genes as treatment for inherited genetic disorders in vivo.
Other features and advantages will be in part obvious and in part pointed out more in detail hereinafter.
These and related advantages are achieved in accordance with the present invention, by providing a new and improved soluble polynucleotide carrier system based on the use of cell surface receptors or binding sites that recognize and internalize certain specific biological compounds or molecules. This internalization process, known as endocytosis, is a normal biological function and the method of the present invention provides a system whereby soluble polynucleotide complexes utilize this specific targeted receptor-mediated endocytotic mechanism.
A better understanding of the features and advantages of the present invention will be obtained from the following detailed description and the accompanying drawing as well as from the illustrative applications of the invention. This includes the several components of the invention and the relation of one or more of such components with respect to each of the others and the process including the several steps thereof and their relationship with each of the others as well as the features, characteristics, compositions, properties and relation of elements described and exemplified herein.